An improved genome editing system for Sphingomonadaceae

Abstract

The sphingomonads encompass a diverse group of bacteria within the Sphingomonadaceae family, with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their capability to degrade or metabolise a variety of recalcitrant organic pollutants. However, the research and development of their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for Sphingopyxis granuli, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious re-streaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting ecfG2, the master regulator of the general stress response in S. granuli. This adds onto the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.